This project seeks to develop novel immunologic therapies for allergic diseases as well as the Laboratory techniques required to understand their mechanism of action and rational application. We have developed a highly sensitive allergen specific T cell cytokine assay which allows us a direct high through put means to examine cytokine responses in clinical trials of allergen immunotherapy. This technique directly measures both the frequency and cytokine profile of antigen specific T cells and provides insight into the mechanism of action of immunomodulatory therapies. Strategies that are being pursued include the administration of rhIL-12 as an adjuvant in allergen immunotherapy as well as studies of the mechanisms of action of traditional immunotherapy. We are applying a similar approach to mouse vaccine models to understand how DNA vaccines generate specific immune responses. To date, we have developed an antigen specific assay utilizing cytometry, allowing the direct detection of complex cytokine phenotypes at the single cell level. We have examined the cytokine response to aeroallergens such as house dust, mite and cat, demonstrating a uniform Th2 response in allergic asthmatics but little or not detectable response in healthy controls. These results suggest that the normal T cell response to ubiquitous aeroallergens is tolerance. A phase I trial using rhIL-12 as an adjuvant in allergen immunotherapy has been approved by the NIAID, IRB and is waiting FDA approval. Antigen specific cytokine flow has been applied to DNA vaccination of mice, demonstrating that DNA vaccination creates a long lasting Th1 polarized immune response in both CD4+ and CD8+ subsets, whereas protein immunization generates a more transient response limited to CD4 T cells.